Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
Front Physiol
2019 Jan 01;10:1334. doi: 10.3389/fphys.2019.01334.
Show Gene links
Show Anatomy links
Expression, Localization, and Effect of High Salt Intake on Electroneutral Na+/HCO3- Cotransporter NBCn2 in Rat Small Intestine: Implication in Intestinal NaCl Absorption.
Wang JL
,
Zhao L
,
Zhu J
,
Wang DK
,
Ren MJ
,
Wang M
,
Liu Y
,
Boron WF
,
Chen LM
.
???displayArticle.abstract???
The electroneutral Na+/HCO3- cotransporter NBCn2 (SLC4A10) of solute carrier family 4 (SLC4) plays important physiological and pathological roles in the body. Our previous study showed that NBCn2 is expressed on the protein level in the small intestine of rat. Here, by reverse-transcription polymerase chain reaction (PCR), we identified a novel full-length NBCn2 variant, i.e., NBCn2-K, from rat small intestine. By pHi measurement with Xenopus oocytes, the activity of NBCn2-K is not significantly different from NBCn2-G. By western blotting, NBCn2 and the Na+/H+ exchanger NHE3 (SLC9A3) are predominantly expressed in the jejunum of rat small intestine. By immunofluorescence, NBCn2 and NHE3 are localized at the apical domain of the jejunum. NaCl overload decreases the expression of NBCn2 by 56% and that of NHE3 by 40% in the small intestine. We propose that NBCn2 is involved in the transepithelial NaCl absorption in the small intestine, and that the down-regulation of NBCn2 by NaCl represents an adaptive response to high salt intake in rat.
FIGURE 1. 5′-RACE and RT-PCR analysis of Slc4a10 expression in small intestine of rat. (A) Diagram of structure of rat Slc4a10 gene. Rat Slc4a10 contains 30 exons and three promoters: promoter P1 (upstream of exon 1), P2 (located in the intron between exons 1 and 2), and P3 (located in the intron between exons 3 and 4). Exon 1 contains the coding sequence for unique Nt of MEIK-NBCn2. Exon 4 contains the coding sequence for the unique Nt of MCDL-NBCn2. Exons 6-26 boxed in gray encode the transmembrane domain of NBCn2. (B) Agrose gel analysis of 5′-RACE of Slc4a10 from small intestine. (C) Agrose gel analysis of RT-PCR product of full-length Slc4a10 transcripts from small intestine. Arrows a, b, and c in panel (A) indicate the approximate positions of the primers used for 5′-RACE. Arrowheads in panel (B) indicate the target bands of PCR products of Slc4a10.
FIGURE 2. Sequence alignment of three partial clones obtained by 5′-RACE. All three clones of C4, C5, and C6 include exon 1 (encoding “MEIK”) and exon 4 (encoding “MCDL”). Clone C4 contains additional 4 nucleotides (nt) “ACAG” at the 5′-end of exon 4, and thus is predicted to express MCDL-NBCn2. Both clones 5 and 6 contain exon 1 and exon 4 in frame, and thus are predicted to express MEIK-NBCn2. Stars indicate the sequences identical in all three clones. The red boxes indicate the coding region of exon 1 for “MEIK⋅⋅⋅⋅⋅⋅”, whereas the blue boxes indicate the coding region of exon 4 for “MCDL⋅⋅⋅⋅⋅⋅”.
FIGURE 3. Diagrams of partial-exon structures of rat Slc4a10 transcripts and full-length variants of NBCn2. (A) Partial-exon structures of Slc4a10 transcripts. (B) Diagrams of predicted primary structures of full-length NBCn2 variants. Not shown here are two full-length variants identified in mouse NBCn2-I and -J with minor variation at the Ct end, as well as four Nt-truncated NBCn2 variants originally identified from rat kidney (Wang et al., 2015). NBCn2-K is predicted to start with “MEIK”. However, the NBCn2 in small intestine can be detected with anti-MCDL, but not with anti-MEIK (see text in “Discussion”).
FIGURE 4. Functional characterization of NBCn2 in Xenopus oocytes. (A) Representative recordings of Vm and pHi of an oocyte expressing rat NBCn2-G. (B) Representative recordings of Vm and pHi of an oocyte expressing rat NBCn2-K. (C) Representative recordings of Vm and pHi of a control oocyte injected with H2O. (D) Summary of pHi recovery rate dpHi/dt. The dpHi/dt of both NBCn2-G and NBCn2-K are not significantly different from each other, but both are significantly higher than that of H2O-injected control oocytes by one-way ANOVA analysis followed by Fisher’s comparison. The numerals in the parentheses in panel D indicate the number of individual oocytes included in each bar.
FIGURE 5. Distribution of NBCn2, NHE3, and Na+-K+ pump in different segments of rat small intestine. (A) Image showing the tissue collection sites for different fragments of the small intestine. S1, duodenum; S2, proximal jejunum; S3, middle jejunum; S4, distal jejunum; S5, ileum. Scale bar, 5 cm. (B) Western blotting of NBCn2 (with anti-MCDL), NHE3, and Na+-K+ pump in segments S1-S5 of the small intestine. β-actin is used as loading control. (C) Fractional distribution of NBCn2 in S1-S5. (D) Fractional distribution of NHE3 in S1-S5. (E) Fractional distribution of Na+-K+ pump in S1-S5. The density of NBCn2 was normalized to that of actin of the same lane from blots like those shown in panel (B). The fractional distribution of NBCn2 in each segment was computed by dividing this normalized density by the sum of the normalized densities of S1-S5. The fractional distribution of NHE3 and Na-K pump was computed by a similar approach. Ns in panels C–E indicate the number of rat individuals included in each panel. One-way ANOVA followed by Fisher’s comparison was used for statistical analysis. P < 0.05 is considered significantly different. Bars not marked by a same alphabet are significantly different from each other.
FIGURE 6. NBCn2 and NHE3 are expressed at the apical membrane of epithelium in small intestine. (A) Overview of staining with anti-MCDL (NBCn2) in the small intestine. (B) Overview of staining with anti-NHE3 in the small intestine. (C) Negative staining of anti-NBCn1 in the small intestine. In these experiments, the final concentrations of anti-MCDL, anti-NHE3, and anti-NBCn1 for immunofluorescence staining were 1.5 μg/ml (1:400 dilution). NBCn2 and NHE3 are mainly expressed in the villi of the small intestine. In panel C, no significant staining was observed for anti-NBCn1 when visualized under microscopy with parameters the same as those used for panel A and C. Inset in panel C shows that the non-specific background staining by anti-NBCn1, when visualized by a much higher exposure, is distributed throughout the cytosol of the epithelia. (D–F) High magnification view shows that NBCn2 is exclusively expressed at the apical membrane of small intestineepithelium. (G–I) High magnification view shows that NHE3 is exclusively expressed at the apical membrane of small intestineepithelium. In these experiments, the basolateral membrane is stained by α1 of Na+-K+ pump.
FIGURE 7. High NaCl intake decreases expression of NBCn2 (A) and NHE3 (B) in small intestine of rat. Membrane preparations of the small intestine were used for western blotting to examine the expression of NBCn2 (probed with anti-MCDL) or NHE3. The density of the target transporter in each lane was normalized to that of actin of the same lane. This ratio of each lane was then normalized to the average of the ratios of the control lanes in the same blot. Compared to the control, NaCl treatment decreases the abundance of NBCn2 by 55% and that of NHE3 by 40%. Numerals in the parenthesis indicate the numbers of rats included in each condition. For statistical comparison, two-tailed student’s t-test was performed. “−” indicates the controls, whereas “+” indicates the rats treated with NaCl.
FIGURE 8. Model to show the hypothetical role of NBCn2 in small intestineepithelium. In the apical membrane, NHEs (e.g., NHE2 and NHE3) in concert with SLC26 transporters mediate NaCl absorption. The NaCl absorption via this pathway involves the action of carbonic anhydrase (CA) in both the luminal side and the cytosol. The presence of NBCn2 in the apical membrane likely provides an alternate pathway for the absorption of Na+ and the direct uptake of luminal HCO3–. In the basolateral membrane, NaCl extrusion is mediated by the coupled action of Na+-K+ pump and Cl– channel ClC-2 (Lipecka et al., 2002; Pena-Munzenmayer et al., 2005). Kir7.1 expressed in the basolateral membrane provides a pathway for the cycling of K+ (Partiseti et al., 1998; Nakamura et al., 1999).
Belengeanu,
A de novo 2.3 Mb deletion in 2q24.2q24.3 in a 20-month-old developmentally delayed girl.
2014, Pubmed
Belengeanu,
A de novo 2.3 Mb deletion in 2q24.2q24.3 in a 20-month-old developmentally delayed girl.
2014,
Pubmed
Böger,
NFAT5 and SLC4A10 Loci Associate with Plasma Osmolality.
2017,
Pubmed
Charney,
Na+-K+-activated adenosine triphosphatase and intestinal electrolyte transport. Effect of adrenal steroids.
1975,
Pubmed
Chen,
Use of a new polyclonal antibody to study the distribution and glycosylation of the sodium-coupled bicarbonate transporter NCBE in rodent brain.
2008,
Pubmed
,
Xenbase
Collins,
Molecular cloning, sequencing, tissue distribution, and functional expression of a Na+/H+ exchanger (NHE-2).
1993,
Pubmed
Damkier,
Na+-dependent HCO3- import by the slc4a10 gene product involves Cl- export.
2010,
Pubmed
Endeward,
Evidence that aquaporin 1 is a major pathway for CO2 transport across the human erythrocyte membrane.
2006,
Pubmed
,
Xenbase
Esposito,
Water absorption and swelling in the rat small intestine in vitro.
1977,
Pubmed
Fordtran,
The mechanisms of sodium absorption in the human small intestine.
1968,
Pubmed
Gawenis,
Electroneutral sodium absorption and electrogenic anion secretion across murine small intestine are regulated in parallel.
2004,
Pubmed
Gawenis,
Intestinal NaCl transport in NHE2 and NHE3 knockout mice.
2002,
Pubmed
Geyer,
Relative CO(2)/NH(3) selectivities of mammalian aquaporins 0-9.
2013,
Pubmed
,
Xenbase
González Bosc,
Effects of atrial natriuretic peptide in the gut.
2000,
Pubmed
Guo,
Na+/HCO3- Cotransporter NBCn2 Mediates HCO3- Reclamation in the Apical Membrane of Renal Proximal Tubules.
2017,
Pubmed
Gurnett,
Disruption of sodium bicarbonate transporter SLC4A10 in a patient with complex partial epilepsy and mental retardation.
2008,
Pubmed
Hilgen,
Lack of the sodium-driven chloride bicarbonate exchanger NCBE impairs visual function in the mouse retina.
2012,
Pubmed
Hoogerwerf,
NHE2 and NHE3 are human and rabbit intestinal brush-border proteins.
1996,
Pubmed
Hubel,
Effect of luminal sodium concentration on bicarbonate absorption in rat jejunum.
1973,
Pubmed
Huebner,
Early Hearing Loss upon Disruption of Slc4a10 in C57BL/6 Mice.
2019,
Pubmed
Humphreys,
Anion effects on fluid absorption from rat jejunum perfused in vivo.
1983,
Pubmed
Jacobs,
Mice with targeted Slc4a10 gene disruption have small brain ventricles and show reduced neuronal excitability.
2008,
Pubmed
Janecke,
Reduced sodium/proton exchanger NHE3 activity causes congenital sodium diarrhea.
2015,
Pubmed
Kandasamy,
Genomic organization and glucocorticoid transcriptional activation of the rat Na+/H+ exchanger Nhe3 gene.
1996,
Pubmed
Kararli,
Comparison of the gastrointestinal anatomy, physiology, and biochemistry of humans and commonly used laboratory animals.
1995,
Pubmed
Kato,
Regulation of electroneutral NaCl absorption by the small intestine.
2011,
Pubmed
Krepischi,
Two distinct regions in 2q24.2-q24.3 associated with idiopathic epilepsy.
2010,
Pubmed
Laforenza,
Aquaporin-6 is expressed along the rat gastrointestinal tract and upregulated by feeding in the small intestine.
2009,
Pubmed
Lipecka,
Distribution of ClC-2 chloride channel in rat and human epithelial tissues.
2002,
Pubmed
Liu,
Cloning and functional characterization of novel variants and tissue-specific expression of alternative amino and carboxyl termini of products of slc4a10.
2013,
Pubmed
,
Xenbase
Liu,
Distribution of NBCn2 (SLC4A10) splice variants in mouse brain.
2010,
Pubmed
,
Xenbase
Matsushita,
Effects of atrial natriuretic peptide on water and NaCl absorption across the intestine.
1991,
Pubmed
Murer,
Sodium/proton antiport in brush-border-membrane vesicles isolated from rat small intestine and kidney.
1976,
Pubmed
Musa-Aziz,
Using fluorometry and ion-sensitive microelectrodes to study the functional expression of heterologously-expressed ion channels and transporters in Xenopus oocytes.
2010,
Pubmed
,
Xenbase
Musa-Aziz,
Relative CO2/NH3 selectivities of AQP1, AQP4, AQP5, AmtB, and RhAG.
2009,
Pubmed
,
Xenbase
Nakamura,
Inwardly rectifying K+ channel Kir7.1 is highly expressed in thyroid follicular cells, intestinal epithelial cells and choroid plexus epithelial cells: implication for a functional coupling with Na+,K+-ATPase.
1999,
Pubmed
Nilsson,
Whole-Genome Sequencing of Cytogenetically Balanced Chromosome Translocations Identifies Potentially Pathological Gene Disruptions and Highlights the Importance of Microhomology in the Mechanism of Formation.
2017,
Pubmed
Parker,
Characterization of human SLC4A10 as an electroneutral Na/HCO3 cotransporter (NBCn2) with Cl- self-exchange activity.
2008,
Pubmed
,
Xenbase
Partiseti,
Cloning and characterization of a novel human inwardly rectifying potassium channel predominantly expressed in small intestine.
1998,
Pubmed
,
Xenbase
Patra,
Bicarbonate enhances sodium absorption from glucose and glycine rehydration solutions. An in vivo perfusion study of rat small intestine.
1989,
Pubmed
Peña-Münzenmayer,
Basolateral localization of native ClC-2 chloride channels in absorptive intestinal epithelial cells and basolateral sorting encoded by a CBS-2 domain di-leucine motif.
2005,
Pubmed
Pilling,
Gene expression markers of age-related inflammation in two human cohorts.
2015,
Pubmed
Potter,
Novel gene function revealed by mouse mutagenesis screens for models of age-related disease.
2016,
Pubmed
Schultheis,
Renal and intestinal absorptive defects in mice lacking the NHE3 Na+/H+ exchanger.
1998,
Pubmed
Sebat,
Strong association of de novo copy number mutations with autism.
2007,
Pubmed
Sinning,
Disruption of Slc4a10 augments neuronal excitability and modulates synaptic short-term plasticity.
2015,
Pubmed
Suzuki,
Expression and localization of aquaporin-1 on the apical membrane of enterocytes in the small intestine of bottlenose dolphins.
2010,
Pubmed
Turnberg,
Mechanism of bicarbonate absorption and its relationship to sodium transport in the human jejunum.
1970,
Pubmed
Turnberg,
Interrelationships of chloride, bicarbonate, sodium, and hydrogen transport in the human ileum.
1970,
Pubmed
Wang,
The Na+-driven Cl-/HCO3- exchanger. Cloning, tissue distribution, and functional characterization.
2000,
Pubmed
,
Xenbase
Wang,
Effects of Nt-truncation and coexpression of isolated Nt domains on the membrane trafficking of electroneutral Na+/HCO3- cotransporters.
2015,
Pubmed
,
Xenbase
Wang,
Identification of an apical Cl(-)/HCO3(-) exchanger in the small intestine.
2002,
Pubmed
,
Xenbase
Woo,
Renal function in NHE3-deficient mice with transgenic rescue of small intestinal absorptive defect.
2003,
Pubmed
Wormmeester,
Quantitative contribution of NHE2 and NHE3 to rabbit ileal brush-border Na+/H+ exchange.
1998,
Pubmed
Xia,
The distinct roles of anion transporters Slc26a3 (DRA) and Slc26a6 (PAT-1) in fluid and electrolyte absorption in the murine small intestine.
2014,
Pubmed
Yun,
Glucocorticoid activation of Na(+)/H(+) exchanger isoform 3 revisited. The roles of SGK1 and NHERF2.
2002,
Pubmed
Zhao,
2q24 deletion in a 9-month old girl with anal atresia, hearing impairment, and hypotonia.
2018,
Pubmed